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Individual preparation of two human T-cell lymphotropic virus type I (HTLV-I) diagnostic GST fused peptides (MTA-1 and GD21) is time-consuming and expensive. The aim of this study was to design a novel single chimeric antigen (SCA) to obviate separate expression of proteins and reduce the cost of reagent preparation.Structural protein fragments, including immunodominant B cell linear epitopes, were selected and different SCAs were designed. Tertiary structure, epitope exposure, solubility and stability were calculated for each SCA and compared with each other. The synthetic DNA  encoding  the  interested  SCA  was  sub-cloned  into  pET32a  expression  vector, expressed as a soluble form in Escherichia coli BL21 (DE3) cells and purified under native condition using affinity chromatography.The SDS-PAGE results indicated that thioredoxin-fused SCA was successfully expressed as a soluble form in E. coli BL21 (DE3) cells. The results of ELISA confirmed that SCA reacted with anti-HTLV-I antibodies in a concentration-dependent manner.Our results indicated that the designed SCA may be a good candidate for the screening of HTLV-I carriers with antigen–antibody-based tests.